Natural killer cell containing exogenous mitochondrium and pharmaceutical composition comprising same

ABSTRACT

An NK cells and PBMC, both having increased cytotoxicity are provided. Particularly, NK cells and PBMC which have exogenous mitochondria introduced thereinto potentiate the immune system of the human body to enhance a therapeutic effect on infectious diseases or cancer. Therefore, the NK cells and PBMC can be used in a composition for prevention or treatment of infectious diseases and cancer. Specially, autogenous NK cells and PBMC guarantee stability without the incurrence of an immune reaction, and thus would be expected to have high commercial activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/349,310, filed on May 13, 2019, which is a National Stage ofInternational Application No. PCT/KR2017/012883, filed on Nov. 14, 2017,which claims priority from Korean Patent Application No.10-2016-0151411, filed on Nov. 14, 2016.

TECHNICAL FIELD

The present invention relates to a cell therapy products, and moreparticularly, to a natural killer (hereinafter referred to as NK) cellor a peripheral blood mononuclear cells (hereinafter referred to asPBMCs) which comprise exogenous mitochondria, and pharmaceuticalcompositions comprising the same as an active ingredient.

BACKGROUND ART

Recently, biopharmaceuticals have been developed to treat variousincurable diseases. The biopharmaceuticals include protein drugs,antibody drugs and cell therapy products. Here, the cell therapyproducts refer to the pharmaceuticals used for the purpose of treatment,diagnosis, or prevention of diseases and the cell therapy products maybe obtained through a series of actions such as by performing ex vivoisolation and proliferation or by altering biological characteristics ofcells by other methods. The cell therapy products may come fromautologous, allogeneic, or xenogeneic cells. Depending on the cell type,the cell therapy products can be categorized into somatic cell therapyproducts and stem cell therapy products.

On the other hand, immunotherapy using the patient's immune function hasbeen paid attention in cancer therapy. In immunotherapy, properties ofimmune cells with diverse functions are utilized and cancer cells areeliminated through complex interactions of the immune cells. A PBMC inhuman blood is a blood cell with a round nucleus such as a lymphocyte ora monocyte, and includes B cells, T cells, macrophages, dendritic cells(DCs), and NK cells. Among them, NK and cytotoxic T lymphocytes (CTLs)cells directly eliminate cancer cells. Antigen-presenting cells thatpresent antigens to these effector cells include dendritic cells or Bcells. In addition, helper T cells and regulatory T cells which secretevarious cytokines, and the like are involved in immune responses. Amongthese, NK cells are emerging and promising immune cells in immune celltherapies.

In particular, as NK cells have been shown to have an ability to killcancer cells in a non-specific manner, many studies have been conductedon NK cells. Based on these researches, NK cell therapies in cancer,have been emerging tools for treatment of cancer cells. In particular,it has been reported that NK cells play an important role in innate andacquired immune responses achieved through cytokine secretion, againstpathogens that have infected a host or cancer.

Accordingly, the present inventors have made efforts to find a newmethod for improving the power of cytotoxicity of NK cells and PBMCs. Asa result, the present inventors have found a method of increasing thecytotoxicity of NK and PBMC, and thus provide a method of treatingcancer using such cytotoxicity, and thus have completed the presentinvention.

Technical Problem

An object of the present invention is to provide an NK cell withincreased cytotoxicity and a pharmaceutical composition comprising thesame.

Another object of the present invention is to provide a PBMC withincreased cytotoxicity and a pharmaceutical composition comprising thesame.

Solution to Problem

In order to achieve the above objects, the present invention provides NKcells comprising exogenous mitochondria, and a pharmaceuticalcomposition for preventing or treating cancer or an infectious disease,comprising the same.

In addition, the present invention provides PBMCs comprising exogenousmitochondria, and a pharmaceutical composition for preventing ortreating cancer or an infectious disease, comprising the same.

Advantageous Effects of Invention

An NK cell and a PBMC, into which exogenous mitochondria have beenintroduced, not only have an increased cytotoxicity which results inincreased cancer-specific killing effects, but also exhibit no sideeffects as immune cells existing in vivo. In addition, the NK cell andthe PBMC are improved in terms of an ability of the cell itself, andthus can be widely applied to various diseases in which the NK cell andthe PBMC are involved. As a result, it is expected that pharmaceuticalcompositions comprising the NK cell and the PBMC are highly commerciallyapplicable.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a result obtained by a PCR analysis method in orderto investigate whether human normal hepatocyte (WRL68)-derivedmitochondria have been delivered into NK cells.

FIG. 2 illustrates a result obtained by FACS analysis in order toinvestigate whether human normal hepatocyte-derived mitochondria havebeen delivered into NK cells.

FIG. 3 illustrates results obtained by fluorescence microscopy, in orderto locate the human normal hepatocyte-derived mitochondria deliveredinto NK cells.

FIGS. 4A and 4B illustrate results showing anti-cancer activity of NKcells into which exogenous mitochondria have been delivered by a CD107adegranulation assay.

FIG. 5 illustrates a result showing the cytotoxic effects of NK cellsinto which exogenous mitochondria have been delivered by K562cytotoxicity assay.

FIG. 6 illustrates a result obtained by FACS analysis in order toinvestigate whether mitochondria derived from umbilical cord mesenchymalstem cells (UC-MSC) have been delivered into NK cells.

FIG. 7 illustrates a result showing the cytotoxic effects of NK cellsinto which UC-MSC-derived mitochondria have been delivered using K562cytotoxicity assay.

FIGS. 8A to 8C illustrate results showing the therapeutic effects by NKcells, into which UC-MSC-derived mitochondria have been delivered in ananimal model of acute myelogenous leukemia, in terms of body weight andsurvival rate of mice.

FIG. 9 illustrates results of expression distribution of blood tumormarkers in an animal model of acute myelogenous leukemia which has beenadministered with NK cells into which UC-MSC-derived mitochondria havebeen delivered.

FIG. 10 illustrates a result showing the cytotoxic effects of PBMCs intowhich exogenous mitochondria have been delivered by K562 cytotoxicityassay

DETAILED DESCRIPTION OF INVENTION

Hereinafter, the present invention will be described in detail.

In an aspect of the present invention, there is provided an NK cellenriched by exogenous mitochondria.

As used herein, the term “exogenous mitochondria” refers to mitochondriaintroduced exogenously rather than mitochondria present in an NK cell.Here, the exogenous mitochondria may be obtained from the same subjectas that from which the NK cell is obtained, but may be obtained fromanother subject. Here, the exogenous mitochondria may be obtained from amammal, and preferably may be obtained from a human. For example, theexogenous mitochondria may be obtained from muscle cells, hepatocytes,fibroblasts, epithelial cells, neurons, adipocytes, osteocytes,leukocytes, lymphocytes, or mucosal cells, and preferably may beobtained from muscle cells with excellent mitochondrial activity. Inaddition, the mitochondria may be obtained from cells cultured ex vivo.

On the other hand, the exogenous mitochondria can be obtained bydisrupting the cells and performing centrifugation, or by culturing thecells, disrupting the cells, and performing the centrifugation. For themethod for obtaining mitochondria, a conventional method used forcollecting an organelle can be used.

Here, an NK cell comprising exogenous mitochondria may be obtained byintroducing mitochondria in an amount of 0.01 to 500 μg, 0.1 to 450 μg,0.5 to 300 μg, 1 to 100 μg, or 2 to 10 μg, per 10⁵ NK cells. Here, an NKcell may contain 1 to 10³ or 10 to 10² exogenous mitochondria.Specifically, the number of exogenous mitochondria may be such thatapproximately 1, 10, 100, or 500 exogenous mitochondria are contained inone NK cell. Here, the number of exogenous mitochondria contained in NKcells can be regulated by controlling an amount of exogenousmitochondria to be introduced into NK cells. Each subject NK cell maycontain a different number of exogenous mitochondria.

In addition, the NK cells may be derived from a mammal or a human.Preferably, the NK cells may be obtained from a subject intended toreceive NK cell therapy. Here, the NK cells may be directly isolatedfrom the blood of the subject and used, or may be obtained bydifferentiating immature NK cells or stem cells obtained from thesubject and used.

Meanwhile, in order to introduce the exogenous mitochondria into NKcells, the exogenous mitochondria and the NK cells may be mixed and thenthe mixture may be subjected to centrifugation so that the mitochondriaare delivered efficiently into the NK cells. A condition at the time ofperforming centrifugation can be appropriately regulated to efficientlyintroduce the mitochondria without damaging the cells. Here, thecentrifugation may be performed at room temperature, and a temperaturecondition can be appropriately selected for cell stability. Here, at thetime of introducing the exogenous mitochondria, the NK cells and theexogenous mitochondria may be mixed in the presence of a surfactant soas to increase membrane permeability of the exogenous mitochondria intothe NK cells, thereby increasing introduction efficiency of theexogenous mitochondria.

Here, the centrifugation may be performed at 100×g, 300×g, 500×g, 800×g,1,000×g, 1,200×g, 1,500×g, 1,800×g, 2,000×g, 2,400×g, 3,000×g, 5,000×g,or 10,000×g. In addition, a centrifugation time may be 0.1 minutes to 60minutes, but is not limited thereto. Specifically, the centrifugationtime may be 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 20minutes, or 30 minutes. In addition, the centrifugation may be performedat a temperature of 0° C. to 40° C., 20° C. to 38° C., or 30° C. to 37°C.

As such, by applying a centrifugal force to both the NK cells and theexogenous mitochondria, the mitochondria can be delivered into the NKcells with high efficiency while causing less damage to the NK cells.

In addition, a surfactant can be used to enhance cell membranepermeability of the exogenous mitochondria into the NK cells. A timepoint at which the surfactant is added may be before, during, or aftermixing of the NK cells with the exogenous mitochondria. In addition,after the surfactant is added to the NK cells, the NK cells may beincubated for a certain period of time in order to increase the cellmembrane permeability of the exogenous mitochondria into the NK cells.An incubation time may be 0.1 to 60 minutes. Specifically, theincubation time may be 1 minute, 5 minutes, 10 minutes, 20 minutes, or30 minutes, but is not limited thereto.

Specifically, the surfactant is preferably a nonionic surfactant, andmay be a poloxamer. Here, the poloxamer is a triblock copolymer composedof a central hydrophobic chain of polyoxypropylene flanked by twohydrophilic chains of polyoxyethylene. In addition, the surfactant inthe mixture may have a concentration of 1 to 100 mg/ml, 3 to 80 mg/ml,or 5 to 40 mg/ml, and may be preferably 10 to 30 mg/ml.

In addition, the method may further comprise a step of incubating themixture under a predetermined time and temperature condition. Theincubation may be performed at a temperature of 0° C. to 40° C., 20° C.to 38° C., or 30° C. to 37° C. In addition, the incubation may beperformed for 0.1 to 4 hours, 0.5 to 3.8 hours, or 0.8 to 3.5 hours. Inaddition, the incubation may be performed for a predetermined time aftercentrifugation is performed so as to deliver the exogenous mitochondriainto the NK cells. In addition, the incubation time can be appropriatelyselected depending on a cell type and an amount of mitochondria.

In another aspect of the present invention, there is provided apharmaceutical composition for treating cancer or an infectious disease,comprising, as an active ingredient, an NK cell that contains theexogenous mitochondria.

Here, the cancer may be any one selected from the group consisting ofgastric cancer, liver cancer, lung cancer, colorectal cancer, breastcancer, prostate cancer, ovarian cancer, pancreatic cancer, cervicalcancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia,brain tumor, neuroblastoma, retinoblastoma, head and neck cancer,salivary gland cancer, and lymphoma. In addition, the infectious diseasemay be any one selected from the group consisting of hepatitis B,hepatitis C, human papilloma virus (HPV) infection, cytomegalovirusinfection, viral respiratory disease, and influenza.

In addition, the pharmaceutical composition may be made into apreparation in liquid or frozen form. Even in a case of being thawedagain after freezing, the pharmaceutical composition does not exhibitimpaired cellular function and can maintain high cell viability andcell-killing ability. Therefore, the pharmaceutical composition can beeasily stored and supplied in a liquid- or frozen-stored form withoutadditional processing.

In still another aspect of the present invention, there is provided amethod for preventing or treating a disease, comprising a step ofadministering, to a subject, a pharmaceutical composition whichcontains, as an active ingredient, NK cells exogenous mitochondria.

Such a method comprises a step of administering an effective amount ofthe NK cell of the invention to a subject having a disease or a subjectsuspected of having a disease. For example, a cell into which exogenousmitochondria has been introduced can be administered to a subject,preferably a mammal, as a therapeutic preparation. The cell can beadministered by an intravenous or subcutaneous route. In a case wherethe composition of the present invention is provided parenterally suchas by intravenous, subcutaneous, ophthalmic, intraperitoneal, orintramuscular route, the composition is preferably in an aqueous form,or it is preferable that the composition includes a physiologicallyapplicable body fluid, suspension, or solution. Accordingly, a carrieror vehicle is physiologically acceptable, and thus can be added to thecomposition and delivered to a patient. Such a carrier or vehicle doesnot adversely affect electrolyte of the patient. Therefore,physiological saline can be generally used as a carrier forpreparations.

The method for preventing or treating a disease using the cell of thepresent invention may also comprise administering another drug orphysiologically active substance having an effect of preventing ortreating the disease, in combination with the cell of the presentinvention. A route, a time, and a dose for the combined administrationcan be determined depending on the type of disease, the patient'sdisease state, the purpose of treatment or prevention, and the otherdrug or physiologically active substance used in combination.

In addition, the disease may be cancer or an infectious disease. Here,the cancer may be selected from the group consisting of gastric cancer,liver cancer, lung cancer, colorectal cancer, breast cancer, prostatecancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroidcancer, laryngeal cancer, acute myelogenous leukemia, brain tumor,neuroblastoma, retinoblastoma, head and neck cancer, salivary glandcancer, and lymphoma. The infectious disease may be any one selectedfrom the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratorydisease, and influenza.

In still yet another aspect of the present invention, there is providedPBMC comprising an exogenous mitochondria.

As used herein, the term “peripheral blood mononuclear cell” refers to acell with a spherical nucleus present in the peripheral blood, which isreferred to as peripheral blood monocyte or PBMC. Such PBMCs may includeimmune cells such as B cells, T cells, macrophages, dendritic cells, andNK cells. The PBMC can be obtained through the blood of a subject. Here,the exogenous mitochondria can be obtained from tissues or cells of thesubject as described above.

Here, the PBMC comprising exogenous mitochondria may be obtained byintroducing mitochondria in an amount of 0.01 to 500 μg, 0.1 to 450 μg,0.5 to 300 μg, 1 to 100 μg, or 2 to 10 μg, per 10⁵ PBMCs. Here, theexogenous mitochondria may be contained in an amount of 1 to 10³ or 10to 100, per one PBMC. In addition, a method of introducing the exogenousmitochondria into the PBMC can be carried out through centrifugation asdescribed above. In still yet another aspect of the present invention,there is provided a pharmaceutical composition for treating cancer or aninfectious disease, comprising, as an active ingredient, the PBMC thatcontains the exogenous mitochondria.

Here, as described above, the cancer may be selected from the groupconsisting of gastric cancer, liver cancer, lung cancer, colorectalcancer, breast cancer, prostate cancer, ovarian cancer, pancreaticcancer, cervical cancer, thyroid cancer, laryngeal cancer, acutemyelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, headand neck cancer, salivary gland cancer, and lymphoma. In addition, theinfectious disease may be any one selected from the group consisting ofhepatitis B, hepatitis C, human papilloma virus (HPV) infection,cytomegalovirus infection, viral respiratory disease, and influenza.

Hereinafter, the present invention will be described in more detail withreference to the following examples. However, the following examples areprovided only to illustrate the present invention, and the scope of thepresent invention is not limited only thereto.

I. Production of NK Cells into which Exogenous Mitochondria have beenIntroduced, and Identification of Functions Thereof Example 1.Production of NK Cells into which Exogenous Mitochondria have beenIntroduced

Human normal hepatocytes (WRL-68) (CRL 1458, ATCC) were seeded inDulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovineserum (FBS; Gibco), 100 μg/ml of streptomycin, and 100 U/ml ofampicillin and cultured for 72 hours. After completion of the culture,the cells were washed twice with Dulbecco Phosphate Buffered Saline(DPBS; Gibco). The washed cells were treated with 0.25% trypsin-EDTA(TE; Gibco) to obtain cells. For the obtained cells, in order to extractmitochondria, a hemocytometer was used to measure the number of cells,and cells in an amount of about 3×10⁶ cells/ml were collected.

Thereafter, the cell line was subjected to primary centrifugation at atemperature of about 4° C. for 10 minutes with a speed of 350×g. Theresulting pellet was collected, and resuspended and homogenized in abuffer solution for 10 to 15 minutes. A composition containing thepellet was subjected to secondary centrifugation at a temperature ofabout 4° C. for 3 minutes with a speed of 1,100×g, to obtainsupernatant. Then, the supernatant was subjected to tertiarycentrifugation at a temperature of about 4° C. for 15 minutes with aspeed of 12,000×g, to isolate mitochondria from the cell line.

The isolated mitochondria were injected, in an amount of 1×10⁵, into atest tube containing separate human NK cells (NK92mi) (CRL2408; ATCC),and centrifugation was performed at a temperature of about 4° C. for 15minutes with a speed of 2,500×g. After removal of supernatant, washingwith PBS was performed and centrifugation was performed at a temperatureof about 4° C. for 5 minutes. Washing was performed twice under the samecondition. Here, the isolated mitochondria were delivered at weights of0.05, 0.05, 0.5, and 5 μg, per 1×10⁵ recipient cells.

Example 2. Identification of Delivery of Human Normal Hepatocyte(WRL68)-Derived Mitochondria into NK Cells (PCR Analysis Method)

A DNA purification kit (NucleoSpin; MACHEREY-NAGEL GmbH & Co. KG) wasused to extract the entire gene from the NK cells collected inExample 1. The extracted DNA was respectively mixed with WRL-68mitochondria-specific identification primers (F: 5′-CTA TTC TCT GTT CTTTCA TGG-3′ (SEQ ID NO: 1), R: 5′-AAG TAT TTA TGG TAC CGT ACG-3′ (SEQ IDNO: 2)). Then, the 2×PCR Master Mix (Applied Biosystems, Foster City,Calif., USA) and tertiary distilled water were added so that a totalvolume of the resultant was made 10 μl, and the Veriti 96-well ThermalCycler (Applied Biosystems) was used to amplify a desired DNA portion.

A PCR reaction was performed to obtain amplified DNA. In order toidentify the amplified DNA, electrophoresis on a 1.5% agarose gel wasperformed, and then staining with Loading Star (DYNEBIO INC., Seongnam,Korea) was performed. A UV-spectrometer (Chemi-Doc XRS; Bio-RadLaboratories, Inc., Hercules, Calif., USA) was used to identifyamplified DNA bands. GAPDH was selected as a house keeping gene. Forthis, primers (F-5′-GGA AGG TGA AGG TCG GAG-3′ (SEQ ID NO: 3), R-5′-GGCAAC AAT ATC CAC TTT ACC-3′ (SEQ ID NO: 4)) capable of amplifying GADPHwere used. This result is illustrated in FIG. 1.

FIG. 1 indicated that an amount of the exogenous mitochondria deliveredinto the NK cells is increased as amounts (0.005, 0.05, 0.5, and 5 μg)of the mitochondria to be mixed with the NK cells are increased.

Example 3. Identification of Delivery of Human Normal Hepatocyte(WRL68)-Derived Mitochondria into NK Cells (FACS Analysis Method)

Fluorescence-activated cell sorter (FACS) analysis was performed toidentify whether hepatocyte-derived mitochondria had been delivered intoNK cells. The mitochondria isolated from human normal hepatocytes weretreated with 500 nM Green mitotracker (Thermo Fisher Scientific,Waltham, USA). The resultant was allowed to react for 10 minutes in a 5%CO2 incubator at 37° C., and washed. The fluorescence-labeledmitochondria derived from hepatocytes were delivered into immune cellsusing a centrifugation method, and then the immune cells wereresuspended in 1 mL of PBS. Then, the mitochondrial delivery wasidentified and analyzed using the FACS Calibur flow cytometer(BDBiosciences, San Jose, Calif., USA). The result is illustrated inFIG. 2.

From FIG. 2, it was identified that the NK cells can be distinguisheddepending on amounts (0.005, 0.05, 0.5, and 5 μg) of the mitochondriadelivered into the NK cells.

Example 4. Identification of Delivery of Human Normal Hepatocyte(WRL68)-Derived Mitochondria into NK Cells (Observation withFluorescence Microscopy)

In order to identify whether normal hepatocyte (WRL-68)-derivedmitochondria had been delivered into human NK cells (NK92mi),mitochondria of the NK cells were treated with 500 nM Green mitotracker(Thermo Fisher Scientific, Waltham, USA), and allowed to react for 10minutes in a 5% CO2 incubator at 37° C. The isolated mitochondria of thehepatocytes were treated with 500 nM red mitotracker. The resultant wasallowed to react for 10 minutes in a 5% CO2 incubator at 37° C., andthen delivered into NK cells. After 5 μg of the mitochondria wasdelivered, the resultant was seeded in a 24-well plate and incubated ina 5% CO2 incubator at 37° C. Then, fluorescence microscopy was used toidentify intracellular delivery within 24 hours. A DAPI reagent fornuclear staining was used as a control staining reagent. The results areillustrated in FIG. 3.

From FIG. 3, it was identified that the exogenous mitochondria had beendelivered into the NK cells.

Example 5. Analysis of Changes in Anti-Cancer Activity of NK Cells intowhich Mitochondria have been Delivered (CD107a Degranulation Assay)

In the human NK cells (NK92mi) collected in Example 1, into whichexogenous mitochondria had been introduced, in order to identifyexpression of CD107a due to degranulation which is an indicator for NKcell activity, the human NK cells and target cells (K562) were mixed ata ratio of 10:1, and then the mixture was treated with fluorescentmaterial-conjugated anti-CD107a. The resultant was co-incubated for 4hours. After the co-incubation, the resultant was treated with anti-CD56for surface staining and allowed to react for 30 minutes. Then,fluorescence-activated cell sorter (FACS) analysis was performed. Theresults are illustrated in FIGS. 4A and 4B.

From FIGS. 4A and 4B, it was identified that anti-cancer activity of theNK cells is increased with amounts (0.05, 0.5, and 5 μg) of theexogenous mitochondria.

Example 6: Identification (K562 Cytotoxicity Assay) of Changes inAnti-Cancer Activity of NK Cells into which Mitochondria have beenDelivered

In order to identify anti-cancer activity of the NK cells collected inExample 1, the collected NK cells were mixed, at 10:1, with target cells(K562) labeled with green fluorescence staining (CFSE; Invitrogen), andthen the mixture was co-incubated for 4 hours in an incubator with acondition of 5% CO₂ at 37° C. After the co-incubation, in order toanalyze the target cells killed by the NK cells, the resultant wastreated with red fluorescence staining (7-AAD; Invitrogen), and thenallowed to react for 10 minutes. Fluorescence intensity of the killedtarget cells was analyzed with fluorescence-activated cell sorter(FACS). The result is illustrated in FIG. 5.

From FIG. 5, it was identified that cytotoxicity against K562 isincreased with amounts (0.05, 0.5, and 5 μg) of the delivered exogenousmitochondria.

Example 7. Production of NK Cells into which Mitochondria of UmbilicalCord-Derived Mesenchymal Stem Cells have been Introduced

Placenta (provided by CHA bundang medical center, IRB No.1044308-201511-BR-022-02)-derived mesenchymal stem cells were seeded inAlpha-Minimum Essential Medium (Alpha-MEM) supplemented with 10% fetalbovine serum (FBS; Gibco), 100 μg/ml of streptomycin, and 100 U/ml ofampicillin, and cultured for 72 hours.

After completion of the culture, the cells were washed twice withDulbecco Phosphate Buffered Saline (DPBS; Gibco). The washed cells weretreated with 0.25% trypsin-EDTA (TE; Gibco) to obtain cells. For theobtained cells, in order to extract mitochondria, a hemocytometer wasused to measure the number of cells, and cells in an amount of about2×10⁷ cells/ml were collected.

Thereafter, the cell line was subjected to primary centrifugation at atemperature of about 4° C. for 10 minutes with a speed of 350×g. Theresulting pellet was collected, and resuspended and homogenized in abuffer solution for 10 to 15 minutes. A composition containing thepellet was subjected to secondary centrifugation at a temperature ofabout 4° C. for 3 minutes with a speed of 1,100×g, to obtainsupernatant. Then, the supernatant was subjected to tertiarycentrifugation at a temperature of about 4° C. for 15 minutes with aspeed of 12,000×g, to isolate mitochondria from the cell line.

The isolated mitochondria were injected, in an amount of 1×10⁵, into atest tube containing separate human NK cells (NK92mi) (CRL2408; ATCC),and centrifugation was performed at a temperature of about 4° C. for 15minutes with a speed of 2,500×g. After removal of supernatant, washingwith PBS was performed and centrifugation was performed at a temperatureof about 4° C. for 5 minutes. Washing was performed twice under the samecondition. Here, the isolated mitochondria were delivered at weights of0.3, 1, 3, 5, and 10 μg, per 1×10⁵ recipient cells.

Example 8. Identification of Delivery of Mitochondria of UmbilicalCord-Derived Mesenchymal Stem Cells (UC-MSCs) into NK Cells (FACSAnalysis Method)

Fluorescence-activated cell sorter (FACS) analysis was performed toidentify whether mitochondria derived from umbilical cord-derivedmesenchymal stem cells had been delivered into NK cells. Mitochondriaisolated from the umbilical cord-derived mesenchymal stem cells weretreated with 500 nM Red mitotracker (Thermo Fisher Scientific, Waltham,USA). The resultant was allowed to react for 30 minutes in a 5% CO2incubator at 37° C. and washed. The fluorescence-labeled mitochondria ofthe umbilical cord-derived mesenchymal stem cells were delivered intoimmune cells using a centrifugation method, and then the cells wereresuspended in 1 mL of PBS. Then, the mitochondrial delivery wasidentified and analyzed using the FACS Calibur flow cytometer(BDBiosciences, San Jose, Calif., USA). The results are illustrated inFIG. 6.

From FIG. 6, it was identified that the NK cells can be distinguishedwith amounts (0.3, 1, 3, 5, and 10 μg) of the UC-MSC-derivedmitochondria which have been delivered into the NK cells.

Example 9. Identification of Changes in Anti-Cancer Activity of NK Cellsinto which Mitochondria Derived from Umbilical Cord-Derived MesenchymalStem Cells (UC-MSCs) have been Delivered (K562 Cytotoxicity Assay)

In order to identify anti-cancer activity of the NK cells collected inExample 7, the collected NK cells were mixed, at 10:1, with target cells(K562) labeled with green fluorescence staining (CFSE; Invitrogen), andthen the mixture was co-incubated for 4 hours in an incubator with acondition of 5% CO₂ at 37° C. After the co-incubation, in order toanalyze the target cells killed by the NK cells, the resultant wastreated with red fluorescence staining (7-AAD; Invitrogen), and allowedto react for 10 minutes. Fluorescence intensity of the killed targetcells was analyzed with fluorescence-activated cell sorter (FACS). Theresults are illustrated in FIG. 7.

From FIG. 7, it was identified that cytotoxicity against K562 isincreased with amounts (0.5, 1, 3, 5, and 10 μg) of the deliveredexogenous mitochondria.

Example 10. Identification of Therapeutic Evaluation on AcuteMyelogenous Leukemia Through Body Weight Change and Survival Rate

6- to 8-week-old male NOD.cg-Prkdcscid IL2rgtm1Sug/JicKoat mice werepurchased from Koatech Co., Ltd. (Gyeonggi-do, Korea). The purchasedmice were subjected to an adaptation period in a clean zone of theexperimental animal center at the CHA University, and then an experimentwas conducted. During the adaptation period, the environment in whichthe mice are kept had day and night at a 12-hour interval, and wasmaintained at a room temperature of 23±2° C. and a humidity of 40% to60%. The mice were subjected to such an adaptation period for 7 days,and then put into the experiment. The mice thus prepared wereadministered, via the tail vein (intravenous (i. v.) injection), K562cells in an amount of 2×10⁵ cells/100 μl, so that an acute myelogenousleukemia model was produced.

Here, mice in which acute myelogenous leukemia had been induced wereadministered, via the tail vein (intravenous (i.v.) injection), the NKcells (NK92mi) prepared according to Example 7 in an amount of 2×10⁶cells/100 μl, so that an experimental group was produced. In the samemanner, a control group was produced by administration of normal NKcells (NK92mi), into which the mitochondria had not been delivered, inan amount of 2×10⁶ cells/100 μl. Body weight change and survival rate ofthe experimental group and the control group were analyzed for 24 daysfrom a time point at which the acute myelogenous leukemia cell line(K562) had been administered. The results are illustrated in FIGS. 8A to8C.

From FIGS. 8A to 8C, it was identified that the group, which has beenadministered the NK cells into which the mitochondria of the umbilicalcord-derived mesenchymal stem cells were delivered, exhibits an about 5%increase in body weight and an about 40% or more increase in survivalrate, as compared with the group which has been administered the normalNK cells into which the mitochondria of the umbilical cord-derivedmesenchymal stem cells were not delivered.

Example 11. Identification of Therapeutic Evaluation on AcuteMyelogenous Leukemia Through Analysis of Tumor-Associated Markers

In order to identify expression distribution of the tumor-associatedmarkers, p53 and c-Myc, the blood of the experimental group and thecontrol group to which an experiment had been conducted according toExample 10 was collected, and then centrifuged at 12,000×g for 15minutes to isolate the serum. The isolated serum was analyzed forexpression distribution of p53 and c-Myc using the Western Blot Kit (WB;Bio-Rad Laboratories, Inc.). The results are illustrated in FIG. 9.

From FIG. 9, it was identified that the group, which has beenadministered the NK cells into which the mitochondria derived from theumbilical cord-derived mesenchymal stem cells were delivered, exhibitsdecreased expression of c-Myc and increased expression of p53, the c-Mycand p53 being blood tumor markers.

II. Production of PBMCs into which Exogenous Mitochondria have beenIntroduced and Identification of Functions Thereof Example 12.Production of PBMCs into which Exogenous Mitochondria have beenIntroduced

The peripheral blood of the inventor which had been taken by a clinician(at CHA medical center) was treated with Ficoll-Paque (AmershamBiosciences) at 1:1 in a Falcon tube, and then the resultant wassubjected to centrifugation at 400×g for 35 minutes, to collect a PBMCpellet. The collected PBMCs were washed twice with PBS. Then, the humanhepatocyte (WRL-68)-derived mitochondria which had been isolatedaccording to Example 1 were delivered into the PBMCs at weights of 0.05,0.05, 0.5, and 5 μg, per 1×10⁵ recipient cells.

Example 13. Identification of Changes in Anti-Cancer Activity of PBMCsinto which Mitochondria have been Delivered (K562 Cytotoxicity Assay)

In order to identify anti-cancer activity of the PBMCs collected inExample 12, the collected PBMCs were mixed, at 10:1, with target cells(K562) labeled with green fluorescence staining (CFSE; Invitrogen), andthen the mixture was co-incubated for 4 hours in an incubator with acondition of 5% CO₂ at a temperature of 37° C. After the co-incubation,in order to analyze the target cells killed by the NK cells, theresultant was treated with red fluorescence staining (7-AAD;Invitrogen), and then allowed to react for 10 minutes. Fluorescenceintensity of the killed target cells was analyzed withfluorescence-activated cell sorter (FACS). The result is illustrated inFIG. 10.

From FIG. 10, it was identified that cytotoxicity against K562 isincreased with amounts (0.005, 0.05, and 0.5 μg) of the deliveredexogenous mitochondria.

1. A peripheral blood mononuclear cell, comprising exogenousmitochondria.
 2. The peripheral blood mononuclear cell of claim 1,wherein the exogenous mitochondria are obtained from muscle cells,hepatocytes, fibroblasts, epithelial cells, neurons, adipocytes,osteocytes, leukocytes, lymphocytes, stem cells, or mucosal cells. 3.The peripheral blood mononuclear cell of claim 1, wherein the exogenousmitochondria are contained in an amount of 1 to 10³ per peripheral bloodmononuclear cell.
 4. The peripheral blood mononuclear cell of claim 1,wherein the exogenous mitochondria are delivered into the peripheralblood mononuclear cell by centrifuging a composition where the exogenousmitochondria and the peripheral blood mononuclear cell are mixed.
 5. Apharmaceutical composition, comprising the peripheral blood mononuclearcell of claim 1 as an active ingredient.
 6. The pharmaceuticalcomposition of claim 5, wherein the composition is in a liquid or frozenform.
 7. A method for treating cancer in a subject in need thereof,comprising a step of administering a pharmaceutical composition thatcontains the peripheral blood mononuclear cell of claim 1 as an activeingredient to the subject.
 8. The method of claim 7, wherein the canceris selected from the group consisting of gastric cancer, liver cancer,lung cancer, colorectal cancer, breast cancer, prostate cancer, ovariancancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngealcancer, acute myelogenous leukemia, brain tumor, neuroblastoma,retinoblastoma, head and neck cancer, salivary gland cancer, andlymphoma.
 9. The method of claim 7, wherein the composition isadministered via a route selected from the group consisting ofintravenous, subcutaneous, ophthalmic, intraperitoneal, andintramuscular routes.
 10. The method of claim 7, which further comprisesadministering another drug or physiologically active substance having aneffect of treating cancer in combination with the cell.
 11. A method fortreating an infectious disease in a subject in need thereof, comprisinga step of administering a pharmaceutical composition that contains theperipheral blood mononuclear cell of claim 1 as an active ingredient tothe subject.
 12. The method of claim 11, wherein the infectious diseaseis selected from the group consisting of hepatitis B, hepatitis C, humanpapilloma virus (HPV) infection, cytomegalovirus infection, viralrespiratory disease, and influenza.
 13. The method of claim 11, whereinthe composition is administered via a route selected from the groupconsisting of intravenous, subcutaneous, ophthalmic, intraperitoneal,and intramuscular routes.
 14. The method of claim 11, which furthercomprises administering another drug or physiologically active substancehaving an effect of treating the infectious disease in combination withthe cell.